Methods and systems for identifying and treating anti-progestin sensitive tumors

ABSTRACT

Methods and systems for identifying and treating a patient suspected of having a tumor susceptible to growth inhibition by anti-progestins are provided. The degree of focal distribution of the progesterone receptor can be used to identify tumors susceptible to treatment with anti-progestin therapy.

This application claims priority to U.S. Provisional Patent No.61/542,931, filed on Oct. 4, 2011, the disclosure of which isincorporated by reference herein in its entirety.

BACKGROUND

The progesterone receptor (PR) is present in cells in two majorisoforms, PR-A and PR-B. In the presence of a bound progestin ligand,such as progesterone, the PR is phosphorylated at specific sites,dimerizes, forms a complex with a number of different cellular elements(e.g., p300 and the steroid receptor coactivator), and binds to specificDNA sequences known as progesterone responsive elements (PREs) toinitiate DNA transcription into RNA. The PR-ligand complex also attractsnumerous other co-activators and co-repressors, which form the cellularelements which in turn transcribe particular genes. These PR complexes(also referred to as foci) can be visualized in the nuclei of cellswhich contain the progesterone receptor as fluorescent aggregates usingimmunohistofluorescence techniques and as dense and dark stained nuclearaggregates using the immunohistochemistry techniques described in thispatent.

In premenopausal women, during the proliferative phase (the first partof the menstrual cycle) when estrogen is the dominant hormone andprogesterone is minimally secreted, staining of normal endometrial cellsfor PR-A and PR-B (e.g., using immunofluorescent techniques and confocalmicroscopy) reveals a diffuse progesterone receptor nuclear stainingpattern. In the secretory phase (the second part of the menstrual cycle)when progesterone is the dominant hormone, using the sameimmunofluorescent techniques and confocal microscopy, staining for PR-Aand PR-B appears as readily detectable fluorescent nuclear foci.

RNA transcription inhibitors have been shown to prevent formation of PRfoci, and 26S proteasome inhibitors have been shown to disrupt the PRnuclear foci. It is therefore believed that the presence of PR foci incells corresponds to active transcriptional complexes, and indicates theactivation of the PR and subsequent gene expression. Conversely, diffusenuclear staining or the absence of PR foci indicates the presence of PRwhich is transcriptionally inactive. Upon exposure of normal breast andendometrium tissues (which are physiologically responsive toprogesterone) to progestin ligands, a change from a diffuse nuclearstaining pattern to focal subnuclear structures can be observed,indicating the activation of the progesterone receptor.

Whereas estrogens are mitogenic (e.g., cause cellular proliferation) fornormal breast epithelial and endometrial cells, the effects ofprogestins are more complex. In the endometrium, progestins inhibitestrogen-induced cell cycle progression early in the G₁ phase, whereasin the breast progestins may both stimulate and inhibit proliferation.In normal breast tissue biopsies it has been shown that proliferativeactivity is stimulated by progesterone (Am J Obstet Gynecol, 1997). Thiscomplexity has led to confounding experimental observations in breastcancer. For example, progestogens appear to have a direct proliferativeeffect on breast cancer cell in vitro when phenol red-free media isused. H. J. Kloosterboer, J. Steroid Biochem. Molec. Biol. Vol. 49, No.4-6, pp. 311-318, 1994. However, when the same contraceptiveprogestogens that induced proliferation in breast cancer cell lines werestudied in an estrogen-dependent DMBA rat breast cancer model, theseprogestogens inhibited tumor progression. Id . . . . It has been shownrecently that many such in vitro experimental models are inadequate.See, e.g., Lange C. et al. Progesterone Receptor Action: TranslatingStudies in Breast Cancer Models to Clinical Insights. Chapter 7 inInnovative Endocrinology of Cancer; 94-111 (2010). Whileprogesterone-induced proliferation has been shown in these experimentalmodels, the majority of proliferating cells were not expressing the PR.Thus, these models do not necessarily predict the efficacy of treatmentwith antiprogestins.

Malignant cells also exhibit nuclear PR foci, but they are different insize and composition from the foci of normal cells. PR foci observed incancer indicate a specific role for the PR which is pertinent to themalignant nature of the cells. For example, the genes activated by thePR in malignant (cancer) breast cells are different than the genesactivated by the PR in normal breast cells; in endometrial cancers PRfoci, but not PR levels, are associated with malignant characteristics;foci in cancer cells are larger, which may be due to alterations in thechromatin remodeling which are common in cancer, and; PR foci in breastcancer are observed regardless of hormonal status (e.g., in the presenceand absence of circulating progesterone in premenopausal andpost-menopausal women respectively). PR foci have been observed (e.g.,using immunofluorescent techniques and confocal microscopy) in the tumorcells of approximately 50% of PR-receptor positive human breast cancerbiopsies. Other patient's tumor samples exhibited a diffuse PR nuclearstaining pattern in the tumor cells using immunofluorescent techniquesand confocal microscopy, indicative of a non-activated or non-functionalform of the PR.

The majority of breast cancers can be treated with hormonal treatments(i.e., anti-estrogens or aromatase inhibitors), which are currently someof the most effective medications used in breast cancer therapy.Hormonal treatment is usually indicated based on the identification ofhormone receptors within the cancer cells. Onapristone (ONA) is ananti-progestin drug which was originally developed for contraceptiveuse. However, it has demonstrated substantial activity in advancedbreast cancer, with a 10% response rate in a study of 101 poor prognosispatients with breast cancer in whom prior hormonal therapy had failed(e.g., breast cancer progressed despite the patient receiving theantiestrogen tamoxifen). In a small breast cancer study using ONA as afirst line hormone treatment, ONA produced a 56% objective responserate, an efficacy in the upper range of the best available treatments inthis disease. ONA binds to the PR, does not induce PR phosphorylationand does not allow the PR to dimerize. The PR-ONA complex binds weakly,or not at all, to its target DNA segment and therefore does not activatethe chromatin remodeling which is a necessary process for DNAtranscription. In in vitro systems, ONA has been shown to reverse the PRnuclear aggregates produced by binding of an artificial ligand to thePR. Gene activation studies have consistently shown that, whileprogestins and other anti-progestins activate progesterone responsivegenes, ONA has minimal activation (i.e., 3 genes).

In addition, ONA is a pure PR antagonist at concentrations which can bephysiologically achieved. ONA does not interfere with other steroidreceptors and does not increase estrogen secretion in human subjects,which is an undesirable side-effect for breast cancer therapy exhibitedby other anti-progestins such as mifepristone.

While onapristone has previously been investigated as a potentialtherapeutic agent for breast cancer, its development was stopped due totoxicity concerns. Robertson et al., Onapristone, a ProgesteroneReceptor Antagonist, as First-line Therapy in Primary Breast CancerEuropean J. of Cancer 35(2) 214-218 (1999). It is important to identifythe subset of the patients with tumors most likely to respond andequally as important to identify the subset of the patients with tumorsleast likely to respond to treatment with ONA and other anti-progestins.Identifying these subsets of patients will allow those patients with APFaccess to a potentially effective cancer treatment and will avoidexposing patients with those cancers for which ONA or otheranti-progestins may not provide benefit to unnecessary toxicity.

Currently, only the presence or absence of the estrogen or progesteronereceptor is considered when making therapeutic decisions on whether touse an endocrine treatment in certain cancers (e.g., breast cancer).Accordingly, conventional assays for PR classify the tumors frompatients with cancer into two categories: PR-positive or PR-negative.One type of assay quantitates the amount of PR per total protein of thecell. These methods can be automated and are quantitative, but are notsatisfactory with respect to accuracy, sensitivity and analysis ofcellular subnuclear receptor structures. A second type of assay includesimmunohistochemical methods using formalin fixed tissue specimens andfluorescent or chromophore labeled monoclonal antibodies targeting thereceptor (either an antibody for each of PR-A and PR-B, or a singleantibody that recognizes both). With immunohistochemical methods, anymicroscopically detectable nuclear staining reaction in more than acertain percentage of cells (typically ≧1%), is reported as being PRpositive as per professional society guidelines. Typically, a clinicalcut off of ≧10% ER or PR positive cells is used to make therapeuticdecisions regarding the use of anti-hormone treatments. No considerationis given to the pattern of cellular or nuclear staining. Relativestaining intensity (i.e., low, medium, or high) is also use as aqualitative measure of hormone receptor positivity. This second type ofassay is more labor intensive and it is not standardized. Typically, lowmagnification microscopic examination is used for the IHC analysis toidentify the presence of the hormone receptor (either estrogen receptor(ER) or PR). Using conventional methods, no analysis of cellulardistribution is done other then an estimate of the percentage of thetumor cells expressing the identified hormone receptor. Analysis of thesubnuclear distribution pattern of the PR requires high poweredmicroscopy. In contrast, high powered microscopy is not needed forstandard IHC determination of hormone receptors in tumor tissue. Theseconventional methods of hormone receptor determination are thus unableto provide information regarding subnuclear PR distribution.

Progestins have complex actions in the breast and other hormonesensitive tissues by targeting distinct cells and having indirecteffects on cells not expressing the PR. PR foci complexes are notqualitatively the same in normal tissue and cancerous tissue, and theydo not necessarily activate the same progesterone receptor associatedgenes. Available clinical data does not fully support the position thatconventional techniques for identifying hormone receptor positive cellsare predictive of anti-hormone efficacy, whether it be for anti-estrogenor anti-progestin directed treatments. Currently, the decision toutilize a hormone treatment (e.g., antiestrogens or aromataseinhibitors) for patients with breast cancer and other hormone sensitivetumors is based on the simple presence of hormone-receptors in tumorsamples. The presence of hormone receptors (ER or PR) does not fullypredict for response to hormone treatment, as only 50-60% ofhormone-receptor positive tumor cases are expected to benefit fromtreatment.

There is a need for a consistent method for predicting the efficacy ofONA and other anti-progestins with respect to heterogeneous “naturallyoccurring” tumors. Further, there is a need for an assay which ispredictive of therapeutic efficacy of ONA and other anti-progestinsagainst the cancers in individual patients.

SUMMARY

An important question pertinent to anti-progestin treatment is how toidentify activated PRs that are relevant clinical therapeutic targets.The present exemplary methods are aimed at characterizing PRs that arepresent in a functional (activated) state in the human tumor tissueroutinely obtainable in the clinical setting. As antagonizing non-activePR with a specific anti-progestin is therapeutically pointless, thepresent methods provide new and critical information to guide treatmentof patients with anti-progestins. Such a predictive diagnostic testwould provide (1) consistent methods to support therapeuticdecision-making with respect to ONA and other anti-progestins, (2) guideselection of individual patients and patient populations that are likelyto respond to treatment, and (3) exclude those individual patients thatare least likely to respond or benefit from an anti-progestin treatment.

In one aspect, a method for identification and treatment of a subset ofprogesterone receptor (PR) positive tumors most susceptible to treatmentwith an anti-progestin such as onapristone (ONA) is provided.Progesterone receptor positive tumors exhibiting a dense, focal PRnuclear distribution pattern, as described herein, are more susceptibleto treatment with anti-progestins such as onapristone. Results from invitro homogeneous, experimental models are not necessarily predictive ofthe properties of naturally-occurring heterogeneous tumors.

In another aspect, a method of inhibiting the growth of a tumorsusceptible to growth inhibition by anti-progestins is provided. Atissue sample suspected of being tumorigenic or cancerous can beobtained from a patient. Progesterone receptor positive cells in thetissue sample can be identified. The degree of distribution of theprogesterone receptor foci in nuclei of the progesterone positive cellsfrom the tissue sample can then be determined and an anti-progestin canbe administered to the patient if the degree of focal distribution inthe tissue sample is greater than about 5% of the progesterone receptorpositive cells.

These patients are more likely to benefit from treatment with ananti-progestin that inactivates activated progesterone foci (APF) (e.g.,ONA) and prevents further formation of APF than patients whose tumors donot express activated PR. The non-activated form of the PR is typicallyseen as diffuse nuclear PR staining. Inactivation of the APF by ananti-progestin may occur by any of a variety of mechanisms, includingdissociation of the foci and inhibition of activation of the fociwithout substantially altering their structure. In one aspect, APFformation can be inhibited or prevented by an anti-progestin throughseveral mechanisms. For example, onapristone may not allow theindividual progesterone receptors to dimerize and prevent the PR frombeing phosphorylated at the ligand phosphorylation sites. The PR-ONAcomplex may bind weakly, or not at all, to its target DNA segment (PREs)and fail to induce the chromatin remodeling which is a necessary processfor DNA transcription. In another example, other anti-progestins mayallow the PR to dimerize and form complexes with co-activators orco-repressors which do not induce DNA transcription.

In this example, DNA binding may occur at the PRE, but transcriptiondoes not occur. Identification of APF may inform the decision of anyanti-progestin treatment as long as the agent interferes with the PRpathway. In one aspect, identification of APF determines the status ofthe PR pathway as activated or not. For example, the use ofmifepristone, or any progestin that complexes with PR and binds to theDNA, could be informed by the identification of APF. The activity ofother agents, including those which would inhibit PR phosphorylation andthus interfere with PR activation, would be predicted by the presence ofAPF in various cancers. Thus, identification of APF could be used toinform treatment recommendations for various classes of compounds whichact by inhibiting the function of the PR.

Patient tumors that do not express activated PR foci (APE) may includethose that are PR-negative by the conventional assay, or those that arePR-positive by the conventional assay. In one aspect, any tumor/cancerwhich exhibits APF is a candidate for treatment with suchanti-progestins, including breast, brain, meningiomas, prostate,ovarian, endometrial, uterine leiomyoma, lung, and uterine cancers.Pulmonary leiomyomatosis which has yet to be formally classified as acancerous condition would also be likely to benefit if APF is expressedin the abnormal tissue. In another aspect, benign tumors not manageablewith standard treatment, but presenting APF, can be treated by anantiprogestin as the presence of APF indicates that the tumor is drivenby aberrant activation of PR, i.e. by the progestin pathway.

Another aspect provides a method of treating patient with a tumorsusceptible to growth inhibition by anti-progestins by obtaining atissue sample suspected of being tumorigenic or cancerous from a patientand exposing the tissue to an anti-progesterone receptor antibody.Progesterone receptor positive cells in the tissue sample can beidentified. The degree of focal binding distribution of the progesteronereceptor in nuclei of cells from the tissue can be determined. If thefocal binding distribution is greater than about 5% of the progesteronereceptor positive cells in the tissue sample, an anti-progestin isadministered to the patient in a dosage range of about 10 to about 200mg per day depending upon the potency, bioavailability, and safetyprofile of the antiprogestin.

In another aspect, the tissue is a specimen of a tumor tissue selectedfrom the group consisting of breast, brain, meningiomas, prostate,ovarian, endometrial, uterine leiomyoma, lung, and uterine tissue.

In another aspect, the presence or absence of focal distribution isdetected by fluorescence, a colorimetic reaction (e.g., an enzymaticreaction), imaged with a counter staining antibody (e.g., chromophore),radioactivity, and Western blot (e.g., differential phosphorylation ofthe PR).

In yet another aspect, the anti-progestin is selected from the groupconsisting of onapristone, lonaprisan, mifepristone, PF-02413873,telapristone, lilopristone, ORG2058, asoprisnil, and ulipristal.

The presence of active progesterone receptor focal distribution isindicated by a degree of nuclear focal distribution of greater thanabout 5% of the progesterone receptor positive cells. In another aspect,a tumor may be heterogeneous with respect to focal distribution andexhibit an active binding pattern (A) with distinct progesteronereceptor foci, a diffuse binding pattern (D) without distinctprogesterone receptor foci, or a mixture of an A pattern and a D pattern(AD) in various areas of the tumor.

In any of the foregoing aspects, when focal distribution (A or ADpattern) is present, the intensity or density of such focal distributionmay be quantitated. For example, progesterone receptor antibodies may beradiolabeled, fluorescently labeled, imaged with a counter stainingantibody (chromophore), imaged with a colorimetic reaction (e.g., anenzymatic reaction), or labeled in another manner where the intensity ofthe label can be measured and quantified.

FIGURES

FIGS. 1A and 1B shows exemplary immunohistochemical brown nuclearstaining patterns in human breast cancer samples derived fromformalin-fixed and paraffin-embedded biopsies using antibodies directedto the progesterone receptor;

FIGS. 2A and 2B show exemplary green nuclear staining patterns in humanbreast cancer samples derived from formalin-fixed and paraffin-embeddedbiopsies using antibodies directed to the progesterone receptor;

FIGS. 3A and 3B show exemplary immunohistochemical brown nuclearstaining patterns with HES background counterstaining in human breastcancer samples derived from formalin-fixed and paraffin-embeddedbiopsies using antibodies directed to the progesterone receptor; and

FIG. 4 shows the percent of breast cancer samples positive for PR-A andPR-B for three binding patterns, A, AD, and D.

DETAILED DESCRIPTION

Before describing several exemplary aspects described herein, it is tobe understood that the invention is not limited to the details ofconstruction or process steps set forth in the following description.The aspects described herein are capable of being practiced or beingcarried out in various ways.

As used herein, the phrases “treating a tumor” and “treatment of atumor” mean to inhibit the replication of tumor cells, inhibit thespread of the tumor, decrease tumor size, lessen or reduce the number oftumor cells in the body, or ameliorate or alleviate the symptoms of thedisease caused by the tumor, decrease the growth of the tumor (increasethe time it takes the tumor to progress) or improve the survival of thepatient when death is due to the cancer or secondary effects of thecancer. The term also includes treatment of cancer. Tumors include bothcancers and non-cancerous tumors. The treatment is consideredtherapeutic if there is a decrease in mortality and/or morbidity,improvement of tumor-related symptoms, or there is a decrease in diseaseburden as may be manifested by reduced numbers of tumor cells in thebody, decreased tumor size or improvement in the time to progression,improvement of progression free survival or improvement of disease freesurvival.

As used herein, the term “APF-active anti-progestin” and its equivalentsrefer to an anti-progestin drug which exhibits an ability to dissolve ordissociate activated PR foci (APF) in the nuclei of cells or inhibit theformation of APF in the nuclei of cells, indicating that its mechanismof action is via the PR activation pathway of the cell.

The terms “APF-positive”, “PR foci positive”, “activated PR”, “PRs in afunctional state” and the like refer to the presence of progesteronereceptor aggregates in the nuclei of cells.

The term “focal distribution” refers to the distribution of “foci”(i.e., aggregation of progesterone receptors) in the nuclei ofprogesterone positive cells. Speckled or hyperspeckled pattern are termsthat can be used referring to steroid nuclear receptor foci pattern inbiology.

The term “degree of focal distribution” refers to the relative amount ofPR foci present in the nuclei of progesterone positive cells. The degreeof focal distribution can be determined quantitatively or qualitatively.

For example, the use of a colorimetric, enzymatic, or radiolabeledligand such as a progesterone receptor antibody, can be used to bind toprogesterone receptors in cell nuclei. The degree of focal distributioncan be determined quantitatively, for example, by measuring colorintensity, fluorescence or quantifying the level of radioactivityemitted by the labeled antibody. The degree of focal distribution candetermined qualitatively by comparing the intensity of binding between acontrol sample and a labeled sample using a light microscope at anappropriate magnification or techniques including, but not limited to,DNA microarray, protein profiling, radiolabeling, or other surrogatesfor measuring APF.

The term “diffuse pattern” refers to a finely granular pattern which isindicative of the absence of focal distribution.

The term “progestin” refers to a natural or synthetic progestationalsubstance that mimics some or all of the actions of progesterone, alsoreferred to as progesterone receptor modulators (PRM) or selectiveprogesterone receptor modulators (SPRM).

The term “anti-progestin” refers to a substance that inhibits theformation, transport, or action of or inactivates progestational agents,including, but not limited to, onapristone, lonaprisan, mifepristone,PF-02413873, telapristone, lilopristone, ORG2058, asoprisnil, andulipristal. A PRM or SPRM may have some anti-progestin properties, andbe considered an anti-progestin or a progestin depending on the contextof use.

The term “antibody” or “antibodies” refers to a protein which is capableof specifically binding to an antigen and includes any substance, orgroup of substances, which has a specific binding affinity for anantigen to the exclusion of other substances. Generally, the term“antibody” includes polyclonal antibodies, monoclonal antibodies,antibodies derived from humans or animals, humanized antibodies (e.g.,non-binding portions derived from a human, binding portions derived froman animals) and fragments thereof.

The terms “anti-PR-A” and “anti-PR-B” antibodies refer to antibodiesdirected to isoforms of the progesterone receptor—PR-A and PR-Brespectively. Anti-PR-AB” refers to an antibody capable of binding toboth PR-A and PR-B. Specific antibodies suitable for use in accordancewith aspects herein include, but are not limited to, PgR636 and PgR1294(M. Press, et al. (Steroids (2002) 67:799-813)), Novacastra clone 16,clone SAN27, clone 1A6, Dako clone PgR636, Ventana, clone 1E2, NovusBiologicals Progesterone Receptor [p Ser 62] Antibody Clone 1064-E2;Novus Biologicals Progesterone Receptor [p Ser190] Antibody CloneEP1516Y, Novus Biologicals Progesterone Receptor [p Ser294] AntibodyClone 608, Abcam Progesterone Receptor [p Ser400] Antibody Ref ab60954,and Genetex Progesterone Receptor [p Ser554] Antibody Ref. GTX118987.

The term “administer” refers to providing a drug or drugs, prescribingone or more drugs, or placing one or more drugs on a formulary. The term“providing” refers to dispensing the drug directly to patient throughany suitable route of administration (e.g., oral, injection,intravenous, intramuscular, and transdermal etc.) or providinginstructions to a patient to do the same.

One aspect provides a method of inhibiting the growth of a tumorsusceptible to growth inhibition by anti-progestins by obtaining atissue suspected of being tumorigenic from a patient and determining thedegree of focal distribution of anti-progesterone receptor in nuclei ofcells from the tissue. If the degree of focal distribution is greaterthan about 5%, an ant-progestin (e.g., onapristone, lonaprisan,mifepristone, PF-02413873, telapristone, lilopristone, ORG2058,asoprisnil, and ulipristal) can be administered to the patient.

While the role of PR, progestins and anti-progestins in breast and othercancers has previously been studied, the results have been inconclusiveleading to difficulties in diagnosing and treating patients. Multiplemodels have shown the numerous and complex interactions of species,strains, cancer type, carcinogens, and tumor environment among otherfactors. Without being bound by theory, the PR may be pathologicallyactivated with altered physiological properties affecting the activationpotential of the ligand resulting in abnormal or uncontrolledstimulation of cell growth and proliferation. However, the most commonlystudied models originate from a small number of original tumors, andtherefore do not accurately represent the physiological variabilitybetween tumor types or the tumors of different patients. That is, thelimited number of cancer models is insufficient to cover the complexityof heterogenic cancers in a human population.

Studies of the formation of PR foci have been used to test compounds fortheir ability to induce PR translocation from the cytoplasm to thenucleus in genetically engineered cell lines. These assays, such as theThermo Scientific PR (Progesterone Receptor) Redistribution® Assay, useimage analysis and fluorescence microscopy to quantitate nuclearaccumulation of PR in the presence of the test compound. In contrast,aspects provided herein are designed for analysis of PR foci in primarytumor tissue, irrespective of the presence of a PR ligand or a drug. Inone aspect, the exemplary methods described herein relate to thepresence of PR foci in the nuclei of cells in naturally-occurring tumorsindicating an anomaly that can be used to predict the efficacy in thatpatient of an anti-progestin that has PR antagonist properties. Inanother aspect, the characterization of constitutively activated PR inthe clinic has now been found to indicate that tumors and cancers aresusceptible to treatment with anti-progestins, including onapristone.

Onapristone, (e.g.,(8S,11R,13R,14S,17S)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(3-hydroxypropyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one)has the following chemical structure:

Other anti-progestins include: progestational3-(6,6-ethylene-17B-hydroxy-3-oxo-17A-pregna-4-ene-17A-YL)propionic acidG-lactones,3-(6,6-ethylene-17.beta.-hydroxy-3-oxo-17.alpha.-pregna-4-ene-17.alpha.-y-1)propionicacid .gamma.-lactone and the following:

Mifepristone(10S,11S,14S,15S,17R)-17-[4-(dimethylamino)phenyl]-14-hydroxy-15-methyl-14-(prop-1-yn-1-yl)tetracyclo[8.7.0.0̂{2,7}.0̂{11,15}]heptadeca-1,6-dien-5-one

Lilopristone(11-beta,17-beta,17(z))-ropenyl);estra-4,9-dien-3-one,11-(4-(dimethylamino)phenyl)-17-hydroxy-17-(3-hydroxy-1-p;11β-[4-(Dimethylamino)phenyl]-17β-hydroxy-17-[(Z)-3-hydroxy-1-propenyl]estra-4,9-dien-3-one

ORG2058(8R,9S,10R,13S,14S,16R,17S)-16-ethyl-17-(2-hydroxyacetyl)-13-methyl-2,6,7,8,9,10,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-one

Lonaprisan(8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-13-methyl-17-(1,1,2,2,2-pentafluoroethyl)-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

Asoprisnil(8S,11R,13S,14S,17S)-11-[4-[(E)-hydroxyiminomethyl]phenyl]17-methoxy-17-(methoxymethyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

Ulipristal(8S,11R,13S,14S,17R)-17-acetyl-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one

PF-24138734-[3-Cyclopropyl-1-(mesylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy,-2,6-dimethylbenzonitrile

In another aspect, focal PR binding provides a more sensitive andpredictive test than currently-used conventional PR assays. Patientsclassified in conventional PR assays as PR-negative as well as thosethat are conventionally PR-positive may test positive for focal PRnuclear binding and therefore be candidates for treatment withanti-progestins such as onapristone. Thus, a patient previouslyidentified as PR negative using previous methods would not have beenconsidered a candidate for treatment with anti-progestins such asonapristone. The presence of PR foci in patients conventionally testedas PR-negative would explain the apparently anomalous result thatonapristone is active in some of these patients. Aspects describedherein will therefore make hormonal treatment potentially available to agreater number of patients with cancer, including potentially thosepatients with breast cancer that are classified as “triple negative”(i.e., negative for estrogen receptor (ER), PR and Her2).

Exemplary suitable immunohistochemical methods for use in aspectsdescribed herein are described by M. Press, et al. (Steroids (2002)67:799-813) and M. Nadji (Anatomic Pathol. (2005) 123:21-27) herebyincorporated by reference in their entirety. By way of example, primarycancer tissue specimens for analysis may be prepared as paraffinsections or fine needle aspiration smears of the cancer tissue as isknown in the art for conventional PR assays. If paraffin sections areused, the paraffin is first melted by heating the slides, and dewaxedwith xylene. Slides are then rehydrated in decreasing grades of ethanoland exposed to an antibody, preferably a monoclonal antibody thatspecifically binds to PR-A, PR-B, or both. Binding of the antibody isthen detected using any one of the methods known in the art fordetection of antibody binding, examples of which are described below.

One exemplary suitable method for detection of binding of an antibody toits target is a colorimetric assay, typically an enzymatic colorimetricassay. One such method employs peroxidase to produce a colored stainvisible under the light microscope. Endogenous peroxidase in the tissuespecimen is blocked using hydrogen peroxide and endogenous biotin isblocked using a biotin-blocking reagent prior to incubation with theantibody or antibodies. If the primary antibody is a mouse antibody, itis subsequently bound to a biotinylated antimouse immunoglobulin.Streptavidin-peroxidase conjugate is added to bind the enzyme to theantibody-target complex. Color is developed by addition ofdiaminobenzidine and cupric sulfate. The tissue specimen may becounterstained with fast green to increase visibility of the peroxidasestain.

Alternatively, a fluorescence method may be used to detect antibodybinding to PR-A, PR-B or both. In this case, a fluorescently-labeledprimary antibody may be bound to the PR target and detected directlyunder a fluorescence microscope. However, a method employing binding ofan unlabeled primary antibody to the PR followed by binding afluorescently-labeled secondary (e.g., antimouse immunoglobulin)antibody to the primary antibody may reduce non-specific fluorescence.Any fluorescent label known for use in immunohistochemical assays may beused in the aspects described herein, for example FITC (fluoresceinisothiocyanate); fluorescein FITC 520 nm green Alexa 488 515 nm greenphycoerythrin PE 565 nm yellow; phycoerythrin-Texas Red ECD 620 nm red;phycoerythrin-cyanine5 PC5 665 nm deep red; Peridinin chlorophyll PerCP670 nm deep red; phycoerythrin-cyanine 5.5 PC5.5 703 nm far red;phycoerythrin-cyanine 7 PC7 755 far red; E allophycocyanin APC 660 nmdeep red; Allophycocyanin-cyanine 7 APC-CY7.

Both monoclonal and polyclonal antibodies may be useful in aspectsdescribed herein. A non-exhaustive list of suitable monoclonalantibodies is described by M. Press, et al. supra, including twoantibodies which are resistant to formalin fixation and paraffinembedding (PgR636 and PgR1294). Specific antibodies suitable for use inaccordance with aspects herein include, but are not limited to, PgR636and PgR1294 (M. Press, et al. (Steroids (2002) 67:799-813)), Novacastraclone 16, clone SAN27, clone 1A6, Dako clone PgR636, Ventana, clone 1E2,Novus Biologicals Progesterone Receptor [p Ser162] Antibody Clone1064-E2; Novus Biologicals Progesterone Receptor [p Ser 190] AntibodyClone EP1516Y, Novus Biologicals Progesterone Receptor [p Ser294]Antibody Clone 608, Abeam Progesterone Receptor [p Ser400] Antibody Refab60954, and Genetex Progesterone Receptor [p Ser554] Antibody Ref.GTX118987.

In one aspect, binding of the antibody to PR is detected by observationof the stained slide under a light microscope or fluorescence microscopeas appropriate. Magnification is typically about 200× or 400× toevaluate, for example, the percentage of cells positive for binding toan antibody. However, to improve sensitivity for detection of APF it maybe desirable to evaluate the slides at 800×-1000× to facilitate study ofsubnuclear structures.

Samples that are apparently PR negative by microscopy may be evaluatedby flow cytometry to detect positive samples below the threshold oflight or fluorescence microscopy. If flow cytometry indicates rarepositive cells, high magnification ×800-×1000 microscopy may be used tostudy subnuclear structures and identify activated progesterone receptorfoci (APF). However, if the positive cells detected by flow cytometryare too rare to be reliably detected by microscopy for analysis of APF,a fluorescence-activated cell sorter (FACS) can be used to separatepositive cells from the cells in suspension based on their fluorescence(e.g., Sony Cell Sorter SH800, Siemens Immulite 2000). As positive cellsare concentrated but not damaged by this process, the reliability andprobability of successfully visualizing APF on subsequent microscopicevaluation is substantially increased.

The presence or absence of APF in individual tumor cell nuclei may bedetected visually under a light or fluorescence microscope, or by anyother appropriate means, such as fluorescence or colorimetricmeasurements. In one aspect, visual means for detection will be used.The results of staining may be quantitated by noting presence or absenceof APF, or by counting the number or percentage of positive cells.Alternatively, specific characteristics of the staining may bequantitated. For example, detection may include notation of whether ornot focal binding in the form of APF is accompanied by diffuse nuclearstaining, quantitation of positive cells by number or percentage, and/orquantitation of intensity or number/density of APF. Quantitation of APFdensity may be determined as the average number of foci/cell, or usingan arbitrary scale (e.g., “few”, “moderate” or “many”). Intensity maysimilarly be determined using an arbitrary scale, e.g., low/medium/highor a numerical scale such as 1-5. In another aspect, the results of theanalysis of the patient's tumor tissue will be compared to positiveand/or negative controls.

In one aspect, a tumor tissue specimen is judged as APF-positive when1-100%, 5-100%, 25-100% or 50-100% of the nuclei of progesteronepositive cells in the specimen exhibit APF. In yet another aspect, thetherapeutic efficacy of an APF-active anti-progestin may also becorrelated with the intensity of APF staining or with the number ordensity of APF, these parameters may also be used to determine thesensitivity of the tumor to treatment with the APF-activeanti-progestin. In general, and without being bound by theory, thesensitivity of a tumor to treatment with APF-active anti-progestin willincrease with increasing number or percentage of positive cells,increasing intensity of APF and/or increasing number of APF in the cellsof the tumor tissue specimen.

In further aspects, methods for determining the sensitivity of a tumorto APF-active anti-progestins may be either manual (e.g., visualdetection using a fluorescence microscope) or they may be automated orsemi-automated using methods for rapid scanning, detection andquantitation of colorimetrically- or fluorescently-labeled tissuespecimens. For example, a fully automated scanning and analysis systemmay be developed and used in certain aspects. While manual selection ofspecific regions of the tumor to be analyzed may be used in one aspect,(e.g., InScape® immunohistochemistry system ((e.g., InScape®immunohistochemistry system (Quest Diagnostics 3 Giralda Farms Madison,N.J. 07940), an automated system for scanning and analysis of APF incell nuclei can be used to provide automated whole-specimen scanning andanalysis of the antigen-specific immunohistochemistry stained specimen.In another aspect, image recognition can be used to create a digitalimage of the entire stained tissue section. An antigen-specific computeralgorithm can be used to analyze the results of the digital imagerepresenting the whole specimen. In yet another aspect, the software canconfigured to distinguish foci from diffuse background staining in thenucleus, and measure fluorescence intensity and size of foci on acell-by-cell or cluster-by-cluster basis, repeating the process for eachcell or cluster over the entire specimen. These automated methods can,in certain aspects, result in improved accuracy by performing a functionthat is not possible manually, with reduced cost. Full automation canalso make the test accessible to non-expert medical centers.

In one aspect, the decision whether to treat the patient based on theresults of the diagnostic assay is based on the number/percentage,intensity and/or density of APF when they are present. Without beingbound by theory, it is anticipated that the efficacy of treatment withan APF-active anti-progestin will increase with increasing number orpercentage of positive cells, increasing intensity of APF and/orincreasing number of APF in the cells of the tumor tissue specimen.Based on these parameters the medical practitioner may also determinethe dosing, timing and length of treatment. Accordingly, another aspectrelates to use of an APF-active anti-progestin for treating anAPF-positive tumor.

The tumor to be identified or treated according to the above methods mayinclude any cancerous or non-cancerous tumor in which APF occur, and inwhich the presence of APF can be determined. Such cancers or tumorsinclude breast cancer, lung, uterine cancer, uterine leiomyoma, ovariancancer, prostate cancer, brain, and angiomas. Benign tumors which can beidentified or treated according to certain aspects include meningiomas,70% of which express PR by conventional analysis.

The APF-active anti-progestin of the foregoing methods may be anyanti-progestin drug having the ability to inactivate APF (for example bydissolving or dissociating the aggregates or preventing formation of APFor forming inactive APF). Such drugs include onapristone (ONA), butothers with a similar mechanism of action are also suitable for use inaspects described herein.

Another aspect provides methods of identifying a tumor susceptible togrowth inhibition by anti-progestins by obtaining a tissue suspected ofbeing tumorigenic or cancerous from a patient and exposing the tissue toan anti-progesterone receptor antibody. Progesterone positive cells inthe tissue sample can be identified. The degree of focal distribution ofthe progesterone receptor in nuclei of the progesterone positive cellsfrom the tissue sample can be determined and an antiprogestin can beadministered to the patient if the degree of focal distribution in thetissue sample is greater than about 5% of the progesterone receptorpositive cells.

In yet another aspect, a method of treating a patient with a tumorsusceptible to growth inhibition by anti-progestins is provided. Themethod comprises obtaining a tissue sample suspected of beingtumorigenic from a patient and exposing the tissue to ananti-progesterone receptor antibody. The progesterone receptor positivecells in the tissue sample can be identified and the focal bindingdistribution of the progesterone receptor in nuclei of cells from thetissue can be determined. If the focal binding distribution is greaterthan 5% A or AD binding pattern of the progesterone receptor positivecells in the tissue sample, an anti-progestin is administered to thepatient in a dosage range of about 10 to about 200 mg per day dependingupon the potency, bioavailability, and safety profile of theanti-progestin.

In another aspect, the degree of focal distribution can be determined bysuitable method as discussed herein including immunochemical,immunofluorescence, DNA microarray, protein profiling, radiolabeling, orother surrogates for measuring APF.

In another aspect, the tumor tissue is selected from the groupconsisting of breast, meningiomas, prostate, ovarian, endometrial,uterine leiomyoma, lung, and uterine tissue.

In yet another aspect, the anti-progestin is selected from the groupconsisting of onapristone, lonaprisan, mifepristone, PF-02413873,telapristone, lilopristone, ORG2058, asoprisnil, and ulipristal.

In another aspect, the degree of focal distribution is determined byidentifying the binding pattern of progesterone receptor in the nucleiof progesterone positive tissue cells. Heterogeneous tumors includecells which may have active progesterone receptor foci or inactiveprogesterone receptor foci. Therefore, there may be cellular regionscontaining active foci as shown by distinct clumps in the cellularnuclei, and cellular regions which exhibit a more diffuse pattern.

For example, FIG. 1 depicts two exemplary binding patterns from brownnuclear staining obtained with anti-progesterone antibodies in humanbreast cancer samples fonnalin-fixed and paraffin-embedded tissuesamples obtained from biopsies of breast cancer patients. FIG. 1A showsa diffuse, granular pattern (D) indicative of cells which are not likelyto be susceptible to treatment with anti-progestins. In contrast, FIG.1B shows a mottled binding pattern (A) indicative of cells which arelikely to be susceptible to treatment with anti-progestins. A mixedpattern exhibits both A and D patterns and is termed AD.

In another aspect, the anti-progesterone antibody is selected from thegroup consisting of anti-PR-A antibody, anti-PR-B antibody, and amixture of anti-PR-A and anti-PR-B antibodies, and bispecific anti-PR ABantibodies.

In yet another aspect, the anti-progestin is administered in an amountfrom 10 to about 200 mg per day depending upon the potency,bioavailability, and safety profile of the anti-progestin. Without beingbound by theory, it is believed that by identifying patients with tumorsthat are susceptible to treatment with progestins, a lower dose of theanti-progestin may be used resulting in a lower risk of toxic sideeffects. Thus, a lower dosage range can be used for patients exhibitinggreater than 5% focal distribution of the progesterone receptor. In oneaspect, the A or AD classification could result in different doses,while D pattern would indicate that treatment with an anti-progestintreatment is not warranted.

In yet another aspect, methods for screening antitumor drugs for theability to inactivate APF are provided. These methods are useful, forexample, to identify additional anti-progestins which may be candidatesfor use in treating of APF-positive tumors according to the methodsdescribed herein. In one aspect, the method provides a method ofscreening a drug candidate for the ability to decrease focaldistribution of the progesterone receptor in the nuclei of progesteronereceptor positive cells in a tumor. At least two tumor tissue specimensfrom the same tumor can be obtained. One tumor tissue specimen can beexposed to a drug candidate. The tumor tissue specimens can then beexposed to anti-progesterone receptor antibodies and the degree of focaldistribution of progesterone receptors in the nuclei of the progesteronereceptor positive cells from the tumor tissue specimens can bedetermined. If the focal distribution of the progesterone receptor inthe tumor tissue specimen exposed to the drug candidate is decreasedcompared to tumor tissue specimens not exposed to the drug candidate,the drug candidate is capable of decreasing focal distribution of theprogesterone receptor in progesterone receptor positive cells of thetumor.

Another aspect provides a system for classifying a tumor susceptible fortreatment with an anti-progestin, comprising a tissue sample and atleast one antibody or antibody binding fragment capable of detecting theprogesterone receptor. The antibody or antibody binding fragment can beused to determine the degree of focal distribution of the progesteronereceptor in the progesterone receptor positive nuclei of cells from atumor tissue specimen. In another aspect, the tumor is susceptible totreatment with an anti-progestin if the degree of focal distribution inthe cell nuclei of the progesterone positive cells is greater than about5%.

In another aspect, detecting a decrease in detectable staining of theAPF is an indication of APF inactivating activity of the antitumor drug.Detecting no substantial decrease in detectable staining of the APF isan indication of lack of APF inactivation of the antitumor drug.

In another aspect, an APF-active anti-progestin may be used incombination with additional hormonal treatment that does not act by anAPF inactivation mechanism (e.g., antiestrogens) to achieve improvedtherapeutic efficacy as compared to either agent alone. Alternatively,an APF-active anti-progestin may be used in combination with one or moreconventional chemotherapeutic agents which are negative for APF activityin the screening assay to achieve improved therapeutic efficacy ascompared to either agent alone (e.g., everolimus, trastuzumab, TM1-D,anti-HER2 drugs, bevacizumab, or chemotherapy with agents such aspaclitaxel, docetaxel, taxanes, doxorubicin, liposomal doxorubicin,pegylated liposomal doxorubicin, anthracyclines, anthracenediones,carboplatin, cisplatin, 5-FU, gemcitabine and cyclophosphamide). Forexample, everolimus is an mTor inhibitor that is indicated incombination with an aromatase inhibitor and may, in the future, beindicated in combination with an anti-progestin.

In yet another aspect, detecting the presence of focal distribution ofthe antibody to progesterone receptors in the nuclei may be used as anindication that the tumor of a patient previously treated with anantitumor drug, which has become resistant to that drug, is stillsensitive to an APF-active anti-progestin such as onapristone. In oneaspect, the method can be adapted to determine whether chemoresistanceof a tumor resulting from previous chemotherapy can be reversed bytreatment with an APF-active anti-progestin. Reversal of suchchemoresistance may be based on the different mechanisms of action ofthe previous chemotherapy and the APF-active anti-progestin.

Another aspect is directed to a system for classifying a tumorsusceptible for treatment with an anti-progestin. The system comprises atissue sample and at least one antibody or antibody binding fragmentcapable of detecting the progesterone receptor wherein the antibody orantibody binding fragment is used to determine the degree of focaldistribution of the progesterone receptor in the nuclei of cells from atumor tissue specimen and wherein the tumor is susceptible to treatmentwith an anti-progestin if the degree of focal distribution is greaterthan about 5%.

EXAMPLE 1

Tumor specimens from patients with breast cancer (invasive ductalcarcinoma) and endometrial cancer were selected from the archives ofOscar Lambret Cancer Center (Lille, France), anatomical pathologicaldepartment. Patients had previously provided consent for the use oftheir tissues for research purposes. Samples of breast or endometrialtumor tissues which had been fixed in 4% formalin fixative and embeddedin paraffin were obtained.

Immunohistochemistry (IHC) was performed on 3-4 μm sections of thearchival breast or endometrial tumor tissues. The sections weredeparaffinized, hydrated and washed in working buffer (0.05 mol/LTris/HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.6, Dako, Denmark, codeS3006). Antigen retrieval was carried out with the Dako Target RetrievalSolution (modified citrate buffer, pH 6.1, Dako, Denmark, code S1699) ina water bath at 98° C. for 20 min. Then, the sections were covered withthe Dako Peroxydase Block solution to block endogenous peroxides at roomtemperature (RT) for 5 min (Dako EnVision® +/HRP Mouse (DAB+) Kit, Dako,Denmark, code K4007), washed and incubated with the primary antibodiesat the appropriate optimal dilutions at RT for 60 min in a humidifiedchamber (Table 1). Following a 5-min. wash with working buffer, the DakoLabelled Polymer (Dako EnVision® +/HRP Mouse (DAB+) Kit, Dako, Denmark,code K4007) was used for the detection of the primary antibody bindingat RT for 30 min. Chromogen (DAB) was then used with Substrate-Batch atroom temperature for 5-10 min and the sections were lightlycounterstained with Gill's hematoxylin.

Negative controls were obtained by substitution of the primaryantibodies with isotype control mouse IgG1 (Table 1) or with antibodydiluent alone (wash buffer negative control) in the immunohistochemicalstaining procedure.

TABLE 1 Antibodies used for immunohistochemistry Antibody against CloneDilutions Host/Isotype Supplier Code PR, A form 16 1:100 (3.6 μg/ml)Mouse IgG1 Novocastra PGR-312-L-CE 1:200 (1.8 μg/ml) PR, B form SAN271:100 (0.4 μg/ml) Mouse IgG1κ Novocastra PGR-B-CE 1:200 (0.2 μg/ml) PR,A/B forms 1A6 1:40 (1.2 μg/ml) Mouse IgG1 Novocastra PGR-L-CE 1:80 (0.6μg/ml) PR, A/B forms 16SAN27 1:100 (2 μg/ml) Mouse IgG1 NovocastraPGR-AB-L-CE 1:200 (1 μg/ml) Negative control DAK-GO1 1:25 (4 μg/ml)Mouse IgG1 Dako X0931 1:100 (1 μg/ml) 1:200 (0.5 μg/ml)

Immunohistochemistry analysis was performed using a Zeiss Axioscopemicroscope, equipped with an Imaging Model ROHS digital camera.Immunoreactive signals were classified as unequivocal brown labeling oftumor cell nuclei. The intensity of labeling was defined as 0 fornegative, + for weak, ++ for moderate and +++ for strong.

EXAMPLE 2

12 breast cancer samples were analyzed with 3 different antibodies and 4methods in IHC. 6 samples could be processed for furtherimmunohistofluorescence (IHF) analysis.

Immunohistofluorescence was performed using a Zeiss fluorescentmicroscope equipped with a CCD camera and Smart Capture software,specific for capture of fluorescent images. IHF was performed on 3-4 μmsections of the archival breast tumor tissues. The sections weredeparaffinized, hydrated and washed in working buffer (0.05 mol/LTris/HCl, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.6, Dako, Denmark, codeS3006). Antigen retrieval was carried out with the Dako Target RetrievalSolution (modified citrate buffer, pH 6.1, Dako, Denmark, code S1699) ina water bath at 98° C. for 20 min. Then, the sections were incubatedwith the primary antibodies at the appropriate optimal dilutions at RTfor 60 min in a black humidified chamber (Table 2). Following a 5-minutewash with working buffer, appropriate secondary antibody conjugated toAlexa Fluor 488 was used for the detection of the primary antibodybinding at RT for 30 min (Anti-mouse IgG (H+L), F(ab′)2, Cell Signaling,USA, code 4408S, dilution 1:1000 ; Anti-rabbit IgG (H+L), F(ab′)2, CellSignaling, USA, code 4412S, dilution 1:1000). All slides were thenwashed and coverslipped using Vectashield® HardSet Mounting Medium(Vector Labs, USA, code H-1400) and stored refrigerate in the dark untilanalysis, to preserve fluorescence. Negative controls were obtained bysubstitution of the primary antibodies with isotype control mouse IgG1or rabbit serum (see IHC table) or with antibody diluent alone (washbuffer negative control) in the immunohistofluorescence stainingprocedure.

All tumor samples were PR Positive for the three different antibodies.However, the analysis of the nuclear pattern was inconclusive in 6 outof 11 PR positive cases with the bispecific A and B antibody (1 case wasPR negative with this antibody only). Six cases were subjected to IHFanalysis with all of the antibodies. In two cases, the IHF procedurecould not be performed with all antibodies because not enough tumortissue remained available. The four cases could be analyzed with the PRB antibody. The IHF analysis with the other antibodies (PRA and PRA+B)was inconclusive in one instance for characterizing the nuclear pattern.The IHF PR nuclear distribution and binding patterns observed wereconcordant with IHC.

Thereafter, a larger sample was analyzed in IHC with the Anti-PR Aantibody, Anti-PR B antibody, or the mixture of both (called thereafterA+B).

75 breast cancers and 25 endometrial cancer samples were processed. Foreach labeled tumor sample, positive focal distribution was defined asthe percentage of labeled tumor cells in the entire tumor tissue,excluding necrotic areas.

The two basic patterns found are presented in FIG. 1. These images showthe staining of tissue samples with anti-PR antibodies using (IHC). FIG.1A shows a brown, finely granular, and diffuse D pattern. FIG. 1B showsa mottled, clumped pattern representing a positive focal binding Apattern. FIG. 2 shows the same samples processed using IHF. FIG. 2Ashows a diffuse D pattern similar to the IHC result in FIG. 2A. FIG. 2Bshows a similar mottled, clumped, focal binding pattern as in FIG. 2B.The diffuse D pattern of FIGS. 1A and 2A are similar to the resultsobtained in gene-engineered cells that express a fluorescent receptorwhen no progesterone or no progesterone-agonist is present(Arnett-Manfield et Al, 2004, 1C Control, 1D, and 1E) and in normalhuman endometrial tissue and in endometrial cancer (Arnett-Manfield etAl, 2004, 1A, 1B, 1C, 1D, 1E, 1F).

The active A pattern observed in formalin fixed, paraffin embedded tumortissue may differ from images obtained in fresh cells. This is expectedbecause formalin-fixation and paraffin embedding tissue will result inchanges to the cellular contents, thereby resulting in a differentpattern of PR. Another difference relative to the research publicationswhich utilized IHF, is related to the method. In the research setting, aconfocal microscope (i.e. using two laser beams) provides highresolution and 3D images; thin slices of tissue samples (e.g., 2microns) are utilized. The IHC pattern results from a chemical reactionthat modifies the cellular content. In contrast for IHC, a traditionalwide-field microscope is used for reading the standard thicker tumorslices (e.g., 4 microns). The IHC technique described results in someloss of resolution.

The IHF technique is less chemically aggressive for tumor tissues, inthat it does not alter the microscopic cellular architecture. IHFrequires specialized, equipment, a pathologist experienced with thetechnique, and is much more time-consuming. IHF cannot be easily coupledwith other pathology analyses such as standard histology that requiresformalin-fixed paraffin embedded tissues. Thus, in one aspect, IHC maybe used as a routine pathological laboratory procedure. In the developedIHC technique used herein, 4 micrometer tissue sections (a commonly usedthickness for routine clinical analysis) were used for all analysis.

FIGS. 3A and 3B are equivalent to FIGS. 1A and 1B with backgroundstaining. The diffuse pattern observed in 5A, or in immunofluorescence,is darkened by the counterstaining. Likewise, 5B demonstrates grossnuclear anomalies. However, the even, diffuse pattern of 5A is stillcharacteristic with 5A with homogeneous nuclei, while 1B translated indysformed nuclei in 5B.

Thus, two basic patterns are found: a diffuse PR nuclear stainingindicating an absence of activated PRs, or and heterogeneous stainingwhere aggregates, called PR foci, can be recognized within the nucleusof the cells. PR foci are larger than elements of a diffuse pattern thatare substantially smaller (see Figures).

EXAMPLE 3

Three categories or phenotypes have been identified for use with aspectsdescribed herein and which are observed at higher magnification (800×).In contrast, standard magnification (400×) is used in for conventionalIHC PR status determination.

Categories (Observed at High Magnification)

D: Diffuse Staining, no PR Foci (e.g., FIG. 1A)

AD: Area associating A and D cells, or heterogeneous distribution of PRfoci with smaller sizes than A.

A Large Foci distributed in an heterogeneous manner (e.g., FIG. 1B)

This classification (D, AD, and A) was evaluated on 100 additional cases(75 breast cancer and 25 endometrial cancer tissue samples). In somecases the samples were positive for one PR isotype and not the other(e.g., positive for PR-A but not for PR-B).

Breast Cancer Samples (61 cases are analyzed for standard PR expression,12 cases were PR negative for all antibodies, 2 cases had missing data).

TABLE 2 Breast Cancer Tumor Cells Positive for Indicated Antibody InPercentages Number of Cases Mean Min Max Anti-PR A + B 54 34% 5% 90%Anti-PR A Alone 51 31% 5% 90% Anti-PR B Alone 52 32% 5% 85% Either A orB * 58 36% 5% 95% * Each antibody gives statistically similar data withthe same average percent (31-36%) of PR Positive cells and varyingwithin the same range (5-95%). * This is a computation that selects thehighest percentage of PR A or PR B, as it was apparent that with theantibodies used, the rate of positive progesterone receptor cells wasnot the same for both antibodies in a same biopsy.

TABLE 3 Endometrial Cancer Cells Positive for Indicated Antibody 25Cases (3 Negative Cases for All PR Antibodies) In Percentages Number ofCases Mean Min Max Anti-PR A + B 19 31% 2% 100%  Anti-PR A Alone 18 21%5% 90% Anti-PR B Alone 18 23% 5% 85% Either A or B * 20   27%% 5% 90%

C. Focal Distribution

The section below describe the frequencies of A, AD, D patterns and N(negative, no PR staining). All cases were analyzed at highmagnification (800×). Two breast cancer cases were not evaluable. Thedata in table 4 demonstrate that the classification varies with theantibody (PRA or PRB) used, and that there is more variability among theantibodies for the AD pattern. This most likely reflects the inherentderegulation of the two PR receptors (A and B) in cancer tissue. Incertain aspects, antibodies targeted at each of the PR isoforms may beused to provide additional information for interpreting the results ofthe analysis. For example, a case may be “D” with an anti-PR A antibodyand “AD” with the second anti-PR B antibody. Based on the laterclassification of “AD”, a treatment with a anti-progestin would bepotentially appropriate. Similarly, a case may be “A” with an antibodyagainst PR A and “AD” with an antibody against PR B, which couldpotentially require a different (higher) dose of the anti-progestinbecause of the greater degree of malignant cell growth indicated by theaberrant PR activity. Conventional IHC methods to determine PR cannotprovide this information because they only indicate the presence orabsence of hormone receptors (i.e., ER and PR). In one aspect, theactivated PR foci pattern based on analysis with 1 or more separateantibodies would provide additional information for analyzing theactivated PR foci pattern.

TABLE 4 PR Focal Distribution for Breast Cancer Cells In Number of casesNumber of cases A AD D Neg Anti-PR A + B 71 4 21 29 17 Anti-PR A Alone67 3 19 29 16 Anti-PR B Alone 69 1 24 17 27 In Percentages % A AD D NegAnti-PR A and B 101% 6% 30% 41% 24% Anti-PR A Alone  99% 4% 28% 43% 24%Anti-PR B Alone 100% 1% 35% 25% 39%

EXAMPLE 4

In the data set outlined in the tables below, a given tumor sample couldbe APF negative for one antibody and APF positive for another and show adifferent APF pattern for one antibody versus the other antibody.However, the results were generally concordant between PR-A and PR-Bantibodies. This concordance is shown on the diagonal of thecross-tabulations that follow below. The concordance between the twosets of conditions is highlighted in the shaded text box of the table.These results illustrate that in certain aspects, more than one antibodywould provide additional information to identify the APF nucleardistribution pattern.

Table 5 below compares the APF patterns with the PR A antibody inrelationship to the PR A+B antibody mixture in the breast cancersamples. A: Aggregated Pattern with large foci, AD: mix of A Cells and Dcells, or heterogeneous medium-medium size foci. D: diffuse pattern orabsence of Activated PR. The columns classify the cases according to theindicated binding pattern using only the PR-A antibody while the rowsclassify the cases using PR-A+PR-B antibodies. The diagonal, highlightedrow shows the number of concordant cases, i.e., cases with the samebinding pattern using both methods. Other cells show discordant results,i.e., cases with different binding patterns for each method.

TABLE 5 Comparison of the APF patterns with PR A versus PR A + B

Table 6: Breast cancer samples: Cross-tabulation of results obtainedwith an anti-PR B antibody (PR B) vs the mixture of anti-PR A andanti-PR B (PR A+B). A: Aggregated Pattern with large foci, AD: mix of ACells and D cells, or heterogeneous medium-medium size foci. D: diffusepattern or absence of Activated PR. The columns classify the casesaccording to the indicated binding pattern using only the PR-B antibodywhile the rows classify the cases using PR-A+PR-B antibodies. Thediagonal, highlighted row shows the number of concordant cases, i.e.,cases with the same binding pattern using both methods. Other cells showdiscordant results, i.e., cases with different binding patterns for eachmethod.

TABLE 6 Comparison of the APF patterns with PR B versus PR A + B

Table 7: Breast cancer samples: Cross-tabulation of results obtainedwith an anti-PR B antibody (PR B) vs an antibody anti-PR A (PR A). A:Aggregated Pattern with large foci, AD: mix of A Cells and D cells, orheterogeneous medium-medium size foci. D: diffuse pattern or absence ofActivated PR. The columns classify the cases according to the indicatedbinding pattern using only the PR B antibody while the rows classify thecases using PR A antibody. The diagonal, highlighted row shows thenumber of concordant cases, i.e., cases with the same binding patternusing both methods. Other cells show discordant results, i.e., caseswith different binding patterns for each method.

TABLE 7 Comparison of the APF patterns with PR A versus PR B

Endometrial Cancer

Similar patterns of PR nuclear distribution are observed in endometrialcancer samples. Importantly, normal fibroblasts were found in biopsysamples and were noted to be PR positive. These normal fibroblasts had aD PR nuclear distribution phenotype indicating that the PR in thesenormal cells were not activated, most likely because the patients arepost menopausal and thus are not producing physiologic levels ofprogesterone. Therefore, the fibroblasts are not exposed to endogenousprogesterone. In contrast, cancer tissue was presenting activated formof PR (APF) even in absence of physiological progesterone as indicatedby the fibroblast pattern.

Table 8: Endometrial cancer samples: Cross-tabulation of resultsobtained with an anti-PR A antibody (PR A) vs the mixture of Anti-PR Aand an antibody Anti-PR B (PR A+B). A: Aggregated Pattern with largefoci, AD: mix of A Cells and D cells, or heterogeneous medium-mediumsize foci. D: diffuse pattern or absence of Activated PR. The columnsclassify the cases according to the indicated binding pattern using onlythe PR A antibody while the rows classify the cases using PR A and PR Bantibodies. The diagonal, highlighted row shows the number of concordantcases, i.e., cases with the same binding pattern using both methods.Other cells show discordant results, i.e., cases with different bindingpatterns for each method.

TABLE 8 Comparison of the APF patterns with PR A versus PR A + B

Table 9: Endometrial cancer samples: Cross-tabulation of resultsobtained with an anti-PR B antibody (PR B) vs the mixture of Anti-PR Aand an antibody Anti-PR B (PR A+B). A: Aggregated Pattern with largefoci, AD: mix of A Cells and D cells, or heterogeneous medium-mediumsize foci. D: diffuse pattern or absence of Activated PR. The columnsclassify the cases according to the indicated binding pattern using onlythe PR B antibody while the rows classify the cases using PR A and PR Bantibodies. The diagonal, highlighted row shows the number of concordantcases, i.e., cases with the same binding pattern using both methods.Other cells show discordant results, i.e., cases with different bindingpatterns for each method.

TABLE 9 Comparison of the APF patterns with PR B versus PR A + B

Table 10: Endometrial cancer samples: Cross-tabulation of resultsobtained with an anti-PR B antibody (PR B) vs an antibody Anti-PR A (PRA). A: Aggregated Pattern with large foci, AD: mix of A Cells and Dcells, or heterogeneous medium-medium size foci. D: diffuse pattern orabsence of Activated PR. The columns classify the cases according to theindicated binding pattern using only the PR B antibody while the rowsclassify the cases using PR A antibody. The diagonal, highlighted rowshows the number of concordant cases, i.e., cases with the same bindingpattern using both methods. Other cells show discordant results, i.e.,cases with different binding patterns for each method.

TABLE 10 Comparison of the APF patterns with PR B versus PR A

In one aspect, the use of antibodies directed to PR-A and PR-B orbi-specific antibodies directed to PR-A and PR-B can be used together toidentify the AD pattern PR nuclear distribution pattern where use of asingle antibody (e.g., PR-A or PR-B) may not identify the AD pattern incertain cases.

In another aspect, the methods disclosed herein describe a PR nuclearpattern in cancer biopsies shown using, for example, IHC, and confirmedusing fresh tissues and MR The diffuse pattern is found in normalcells/tissues that are not exposed to progestins under experimental andphysiological conditions. The diffuse nuclear distribution patternindicates that the PR of the tumor cells is not activated, and thereforetreatment of the tumor with an antiprogestin is unlikely to beeffective. In contrast, the presence of the AD or A pattern is similarto what is observed when experimental models or normal cells are exposedto progestins. These patterns signal that PRs are activated andtranscriptionally activate in some cells and that treatment withantiprogestins is likely to be effective in these cases.

Expression of these patterns (e.g., A and AD) is heterogeneous in tumorsand across different samples, which is a characteristic of cancers. Incontrast, the D phenotype is homogeneous, a pattern consistent with alack of PR biologic function. The expression of PR and the phenotype wehave described vary according to the expressed PR Isotype (A or B) andthe antibody used (e.g., bispecific AB, A only, B only and the mixtureof A+B). This variability of the PR nuclear distribution pattern is notunexpected in naturally occurring human cancers which are inherentlyheterogenous .

EXAMPLE 5

The plot of FIG. 4 shows the percent of breast cancer samples positivefor PR-A and PR-B for the three binding patterns, A, AD, and D. Theresults support the conclusion that a positive progesterone receptorstatus determined by conventional methods does not correlate with thepresence of PRF distribution as described herein.

EXAMPLE 6

TABLE 11 Table 11 Percentage of Cells expressing the APF pattern withdifferent antibodies PR A + B PRA PRB % of Cells % of Cells % of CellsAPR with APF APR with APF APR with APF AD 30% AD AD A AD  5% AD D D AD 5% AD AD  0% AD 20% D AD  0% AD 10% AD 10% D AD A AD 15% A A AD 40% AD20% A A AD 10% A AD 20% AD 20% AD 50% D D AD  5% AD D AD  5% D D AD 70%A AD 40% AD  5% D AD AD 10% Neg AD 40% AD 60% D AD 10% AD 30% AD D A AD20% D AD AD 20% AD AD 50% AD 30% AD 20% A AD 20% AD 20% A A AD  5%

Table 11 shows the percentage of “A” binding pattern cells for tissuesamples exhibiting both “A’ and “D” binding pattern cells. The columnlabeled “APR” indicates the overall pattern observed for the tissuesample while the “A %” column indicates the percentage of cells in thesample that exhibit the “A” binding pattern. Each row shows the resultsfor one case using both anti-PR-A and anti-PR-B antibodies or eachantibody alone.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It will be apparent to those skilled in the art thatvarious modifications and variations can be made to the method andsystems described herein without departing from the spirit and scope ofthe invention. Thus, it is intended that the present invention includemodifications and variations that are within the scope of the appendedclaims and their equivalents.

1. (canceled)
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 21. A method of inhibiting the growth of a tumor susceptibleto growth inhibition by anti-progestins, comprising: a) obtaining atissue sample suspected of being tumorigenic or cancerous from apatient; b) identifying progesterone receptor positive cells in thetissue sample; c) determining the degree of focal distribution of theprogesterone receptor in nuclei of the progesterone positive cells fromthe tissue sample and identifying the antibody binding pattern of theprogesterone positive cells of tissue; and d) administering ananti-progestin to the patient if the degree of focal distribution in thetissue sample is greater than about 5% of the progesterone receptorpositive cells.
 22. The method of claim 1, further comprisingdetermining the degree of focal distribution in nuclei in cells from thetissue by exposing the tissue to anti-progesterone receptor antibodies.23. The method of claim 1, wherein the tumor is selected from the groupconsisting of breast, brain, meningiomas, prostate, ovarian,endometrial, uterine leiomyoma, lung, and uterine tissue.
 24. The methodof claim 1, wherein the binding pattern is selected from groupconsisting of diffuse (D), active (A), and active/diffuse (AD).
 25. Themethod of claim 2, wherein at least two antibodies are used to determinethe degree of focal distribution.
 26. The method of claim 2, wherein theantibodies are selected from the group consisting of anti-PR-A antibody,anti-PR-B antibody, and a mixture of anti-PR-A and anti-PR-B antibodies,and bi-specific antibodies related to PR-A and PR-B.
 27. The method ofclaim 2, wherein the degree of focal distribution of the progesteronereceptor is determined by a detection method selected from the groupconsisting of immunohistochemistry, immunofluorescence, and Westernblot.
 28. The method of claim 1, wherein the anti-progestin isadministered to the patient in an amount from about 10 mg to about 200mg per day.
 29. A method of treating a patient with a tumor susceptibleto growth inhibition by anti-progestins, comprising: a) obtaining atissue sample suspected of being tumorigenic from a patient; b) exposingthe tissue to an anti-progesterone receptor antibody; c) identifyingprogesterone receptor positive cells in the tissue sample andidentifying the binding pattern of the progesterone positive cells ofthe tissue; d) determining the focal binding distribution of theprogesterone receptor in nuclei of cells from the tissue and identifyingthe binding pattern of the progesterone positive cells of the tissue,wherein if the focal binding distribution is greater than 5% of theprogesterone receptor positive cells in the tissue sample with an A orAD binding pattern, an anti-progestin is administered to the patient ina dosage range of about 10 to about 200 mg per day.
 30. The method ofclaim 9, wherein the tumor is selected from the group consisting ofbreast, brain, meningiomas, prostate, ovarian, endometrial, uterineleiomyoma, lung, and uterine tissues.
 31. The method of claim 9, whereinthe binding pattern is selected from group consisting of diffuse (D),active (A), and active/diffuse (AD).
 32. The method of claim 11, whereinthe antibody is selected from the group consisting of anti-PR-Aantibody, anti-PR-B antibody, a bi-specific antibody directed to PR-Aand PR-B, and a mixture of anti-PR-A and anti-PR-B antibodies.